Paramyxoviruses are among the most common organisms in acute respiratory tract infections, and these ubiquitous pathogens are responsible for a high degree of morbidity and mortality worldwide. These RNA viruses have evolved mechanisms that control both the level and the particular type of viral RNA synthesized during an infection. cis-acting sequences contained within the paramyxovirus genome are a major control point for regulating RNA synthesis. The proposed work will test several new concepts that have emerged concerning features of the viral nucleocapsid template which regulate genome replication and gene expression for the prototypic paramyxovirus SV5. The first aim is to determine the role in viral RNA replication of three factors: (i) sequence within two essential and discontinuous promoter elements, (ii) the spacing of these elements along one face of a helix and (iii) the hexameric phase in the promoter region. A reverse genetics system based on model RNA minigenome analogs will be used along with site specific mutagenesis and an in vivo selection method to determine the relative contribution of each of these factors to promoter activity. Chimeric RNA genomes containing exchanges between the genomic and antigenomic promoters will be used along with polymerase binding assays to determine the basis for differential synthesis of viral RNA from these two promoters. For some paramyxoviruses, there is a very high level of transcriptional readthrough at the junction between the viral M and F genes. In the second aim, a transcription-competent model dicistronic RNA genome will be used to determine the molecular basis for transcriptional readthrough at the M-F junction. In the third aim, recombinant viruses containing altered M-F intergenic regions will be isolated from a full length cDNA. These mutant viruses will be employed to test the two hypotheses that during the course of an infection, M-F readthrough transcription serves to (i) downregulate the level of fusion-competent F protein or (ii) increase the number of polymerase molecules that can access the 5' end of the viral genome. These experiments will test new concepts on how the paramyxovirus nucleocapsid-associated RNA template can modulate the activities of the multifunctional viral polymerase. Results from the proposed work will form a critical base for a rational reverse-genetics approach to parainfluenza vaccines, since it may be possible to design ideal attenuated recombinant viruses which have altered promoter or intergenic transcription activities.